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| Cloning and Characterization Analysis of Raffinose Synthase Gene (TaRS) in Wheat (Triticum aestivum) |
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Abstract Abstract Raffinose is ubiquitously occurred in higher plant as an important soluble carbohydrate, which is known to be involved in various abiotic stresses. Raffinose synthase (RS, EC2.4.1.82) is the key enzyme that catalyses the reversible galactosylation, yielding raffinose and myo-inositol. In this study, the raffinose synthase gene TaRS of wheat (Triticum aestivum) was cloned by homologous alignment. The TaRS was assigned to wheat chromosome 3B. Sequence analysis revealed that TaRS had a whole open reading frame (ORF, 2 349 bp) and belonged to the glycoside hydrolase super family (GH-D) which contained 2 conservative motifs, KxD and RxxxD. In evolutionary relationship, TaRS shared the highest homology with the indicated RS from Aegilops tauschii (XM020298559.1). Southern blot assay showed that there existed at least 4 allelic copies of TaRS in Chinese spring wheat genome. Subcellular localization analysis revealed that TaRS protein was localized at the cell membrane in wheat protoplasts. Tissue specific analysis showed that TaRS expressed in root, stem, leaf and seed, but had the highest expression level in leaves. Heterologous expression was conducted in Escherichia coli, and the crude extract was able to utilize sucrose and galactinol as substrates to synthesis raffinose in vitro based on the result of high performance liquid chromatography (HPLC). Furthermore, TaRS displayed an optimum activity at about pH 8.0. Expression pattern analysis under multiple abiotic stresses indicated that the expression of TaRS was induced by dehydration, high temperature, salinity, and low temperatur, and reached the highest expression level at 12, 1, 1, 48 h, respectively. These results indicated that TaRS may play critical roles in wheat tolerance of abiotic stresses and provides theoretical basis for further studying in wheat abiotic stress tolerance breeding.
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Received: 22 February 2017
Published: 20 July 2017
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| [1]Aker J, Borst J W, Karlova R .The Arabidopsis thaliana, AAA protein CDC48A interacts in vivo with the somatic embryogenesis receptor-like kinase 1 receptor at the plasma membrane[J].Journal of Structural Biology, 2006, 156(1):62-71
[2]Bachmann M, Matile P, Keller F .Metabolism of the Raffinose Family Oligosaccharides in Leaves of Ajuga reptans L. (Cold Acclimation, Translocation, and Sink to Source Transition: Discovery of Chain Elongation Enzyme)[J].Plant Physiology, 1994, 105(105):1335-1345
[3]Bl?chl A, Peterbauer T, Richter A.Inhibition of raffinose oligosaccharide breakdown delays germination of pea seeds[J].Journal of Plant Physiology, 2007, 164(8):1093-1096
[4]Brenac P, Horbowicz M, Downer S M .Raffinose accumulation related to desiccation tolerance during maize ( Zea mays, L.) seed development and maturation[J].Journal of Plant Physiology, 1997, 150(4):481-488
[5]Brenac P, Horbowicz M, Smith M E.Raffinose and Stachyose Accumulate in Hypocotyls during Drying of Common Buckwheat Seedlings[J].Crop Science, 2013, 53(4):1615-1625
[6]Egert A, Keller F, Peters S .Abiotic stress-induced accumulation of raffinose in Arabidopsis leaves is mediated by a single raffinose synthase ( RS5, At5g40390)[J].BMC Plant Biology, 2013, 13(1):285-288
[7]Elsayed A I, Rafudeen M S, Golldack D .Physiological aspects of raffinose family oligosaccharides in plants: protection against abiotic stress[J].Plant Biology, 2014, 16(1):1-8
[8]Findling S, Zanger K, Krueger S .Subcellular distribution of raffinose oligosaccharides and other metabolites in summer and winter leaves of Ajuga reptans, (Lamiaceae)[J].Planta, 2015, 241(1):229-41
[9]Horbowicz M, Obendorf R L .Seed desiccation tolerance and storability: Dependence on flatulence-producing oligosaccharides and cyclitols—review and survey[J].Seed Science Research, 1994, 4(4):385-405
[10]Knaupp M, Heyer A G .Evidence for a role of raffinose in stabilizing photosystem II during freeze-thaw cycles[J].Planta, 2011, 234(3):477-86
[11]Les?aw B, Lahuta, Ryszard J, et al. Raffinose in seedlings of winter vetch ( Vicia villosa, Roth.) under osmotic stress and followed by recovery[J].Acta Physiologiae Plantarum, 2011, 33(3):725-733
[12]Li S, Li T, Kim W D 2007.Characterization of raffinose synthase from rice ( Oryza sativa, L. var. Nipponbare)[J].Biotechnology Letters, 2007, 29(4):635-640
[13]Mieke V, Natalia D, Eveline L.Metabolism of galactosyl-oligosaccharides in Stellaria media--discovery of stellariose synthase, a novel type of galactosyltransferase[J].Phytochemistry, 2010, 71(10):1095-1103
[14]Muzquiz M, Burbano C, Pedrosa M M 1999.Lupins as a potential source of raffinose family oligosaccharides : Preparative method for their isolation and purification[J].Industrial Crops & Products, 1999, 9(3):183-188
[15]Nishizawa A, Yabuta Y, Shigeoka S.Galactinol and raffinose constitute a novel function to protect plants from oxidative damage[J]. Plant Physiology, 147(3):1251-63.[J].Plant Physiology, 2008, 147(3):1251-1263
[16]Osumi, C., Nozaki, J., Kida, T .Methods of producing nucleic acids coding for proteins have raffinose synthase activity[J].US, 2005, US 20050066391 A1(US 20050066391 A1):1-7
[17]Peterbauer T, Mach L, Mucha J .Functional expression of a cDNA encoding pea ( Pisum sativum, L. raffinose synthase, partial purification of the enzyme from maturing seeds, and steady-state kinetic analysis of raffinose synthesis[J].Planta, 2002, 215(5):839-846
[18]Peterbauer T, Richter A .Biochemistry and physiology of raffinose family oligosaccharides and galactosyl cyclitols in seeds[J].Seed Science Research, 2001, 11(3):185-197
[19]Peters S, Mundree S G, Thomson J A.Protection mechanisms in the resurrection plant Xerophyta viscosa (Baker): both sucrose and raffinose family oligosaccharides (RFOSs) accumulate in leaves in response to water deficit[J].Journal of Experimental Botany, 2007, 58(8):1947-19
[20]Roman G, Raimund T .Raffinose Family Oligosaccharides Act As Galactose Stores in Seeds and Are Required for Rapid Germination of Arabidopsis in the Dark[J].Frontiers in Plant Science, 7(7):111., 2016, 7(7):111-111
[21]Salerno G L, Pontis H G .Raffinose Synthesis in Chlorella vulgaris Cultures after a Cold Shock[J].Plant Physiology, 1989, 89(2):648-651
[22]Santos T B, Budzinski I G, Marur C J .Expression of three galactinol synthase isoforms in Coffea arabica L. and accumulation of raffinose and stachyose in response to abiotic stresses[J].Plant Physiology & Biochemistry, 2011, 49(4):441-448
[23]Sauer N, Stolz J .SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker[J].Plant Journal, 1994, 6(1):67-77
[24]Sengupta S, Mukherjee S, Basak P.Significance of galactinol and raffinose family oligosaccharide synthesis in plants[J].Frontiers in Plant Science, 6:656., 2015, 6(6):656- 656
[25]Stadler R, Wolf K, Hilgarth C .Subcellular localization of the inducible Chlorella HUP1 monosaccharide-H+ symporter and cloning of a Co-induced galactose-H+ symporter[J].Plant Physiology, 1995, 107(1):33-41
[26]Sui X L, Meng F Z, Wang H Y.Molecular cloning, characteristics and low temperature response of raffinose synthase gene in Cucumis sativus L. [J].Journal of Plant Physiology, 2012, 169(18):1883-1891
[27]Sun Z, Qi X, Wang Z .Overexpression of TsGOLS2, a galactinol synthase, in Arabidopsis thaliana, enhances tolerance to high salinity and osmotic stresses[J].Plant Physiology & Biochemistry, 69C(8):82-89., 2013, 69C( 8):82-89
[28]Van d E W, Coopman M, Vergauwen R.Presence of Inulin-Type Fructo-Oligosaccharides and Shift from Raffinose Family Oligosaccharide to Fructan Metabolism in Leaves of Boxtree (Buxus sempervirens)[J].Frontiers in Plant Science, 2016, 7(e6605):209-209
[29]Wu X, Kishitani S, Ito Y.Accumulation of raffinose in rice seedlings overexpressing OsWRKY11 in relation to desiccation tolerance[J].Plant Biotechnology, 26(4):431-434., 2009, 26( 4):431-434
[30]Xue H, Chen X, Li G.Involvement of phospholipid signaling in plant growth and hormone effects[J].Current Opinion in Plant Biology, 10(5):483-489., 2007, 10(5 ):483-489
[31]Zhuo C, Wang T, Lu S .A cold responsive galactinol synthase gene from Medicago falcata (MfGolS1) is induced by myo-inositol and confers multiple tolerances to abiotic stresses[J].Physiologia Plantarum, 149(1):67–78., 2013, 149(1 ): 67-78
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